Folding Nucleus Research Articles

Effects of Ionic Salts in Aqueous Environments on the Folding Dynamics of the 21-30 Fragment of the Amyloid Beta-Protein

The amyloid β-protein (Aβ) has been implicated in the pathogenesis of Alzheimer's disease. Although little is known about the initial deleterious misfolding of the full-length Aβ, in vitro experiments have shown that a 21 through 30 fragment of Aβ, the Aβ(21-30), may be the folding nucleus of the full-length protein. Our previous all-atom molecular dynamics work shed light on the behavior of the Aβ(21-30) in bulk water. Here, we explore the effects on the folding dynamics of the Aβ(21-30) of dissolved ionic salts (NaCl, CaCl2, and KCl) that are common to the cellular environment. Using microsecond-long all-atom molecular dynamics we find that previously found extended beta secondary structures are suppressed by KCl, promoted by CaCl2, and slightly promoted by NaCl. Measurements of distances, radial distributions, and contacts of ions surrounding the Aβ(21-30) are also analyzed. Our results suggest that electrostatic interactions between the ions and residues play a less significant role than interactions between solvent and residues due to structure ordering of solvent molecules by local ions.

Conformational Dynamics of Insulin

We have exploited a prandial insulin analog to elucidate the underlying structure and dynamics of insulin as a monomer in solution. A model was provided by insulin lispro (the active component of Humalog®; Eli Lilly and Co.). Whereas NMR-based modeling recapitulated structural relationships of insulin crystals (T-state protomers), dynamic anomalies were revealed by amide-proton exchange kinetics in D2O. Surprisingly, the majority of hydrogen bonds observed in crystal structures are only transiently maintained in solution, including key T-state-specific inter-chain contacts. Long-lived hydrogen bonds (as defined by global exchange kinetics) exist only at a subset of four α-helical sites (two per chain) flanking an internal disulfide bridge (cystine A20–B19); these sites map within the proposed folding nucleus of proinsulin. The anomalous flexibility of insulin otherwise spans its active surface and may facilitate receptor binding. Because conformational fluctuations promote the degradation of pharmaceutical formulations, we envisage that “dynamic re-engineering” of insulin may enable design of ultra-stable formulations for humanitarian use in the developing world.

Molecular chaperone function of Mia40 triggers consecutive induced folding steps of the substrate in mitochondrial protein import

Several proteins of the mitochondrial intermembrane space are targeted by internal targeting signals. A class of such proteins with α-helical hairpin structure bridged by two intramolecular disulfides is trapped by a Mia40-dependent oxidative process. Here, we describe the oxidative folding mechanism underpinning this process by an exhaustive structural characterization of the protein in all stages and as a complex with Mia40. Two consecutive induced folding steps are at the basis of the protein-trapping process. In the first one, Mia40 functions as a molecular chaperone assisting α-helical folding of the internal targeting signal of the substrate. Subsequently, in a Mia40-independent manner, folding of the second substrate helix is induced by the folded targeting signal functioning as a folding scaffold. The Mia40-induced folding pathway provides a proof of principle for the general concept that internal targeting signals may operate as a folding nucleus upon compartment-specific activation.

Misfolding of CasBrE SU is reversed by interactions with 4070A Env: implications for gammaretroviral neuropathogenesis

BackgroundCasBrE is a neurovirulent murine leukemia virus (MLV) capable of inducing paralytic disease with associated spongiform neurodegeneration. The neurovirulence of this virus has been genetically mapped to the surface expressed subunit (SU) of the env gene. However, CasBrE SU synthesized in the absence of the transmembrane subunit (TM) does not retain ecotropic receptor binding activity, indicating that folding of the receptor binding domain (RBD) requires this domain. Using a neural stem cell (NSC) based viral trans complementation approach to examine whether misfolded CasBrE SU retained neurovirulence, we observed CasBrE SU interaction with the "non-neurovirulent" amphotropic helper virus, 4070A which restored functional activity of CasBrE SU.ResultsHerein, we show that infection of NSCs expressing CasBrE SU with 4070A (CasES+4070A-NSCs) resulted in the redistribution of CasBrE SU from a strictly secreted product to include retention on the plasma membrane. Cell surface cross-linking analysis suggested that CasBrE SU membrane localization was due to interactions with 4070A Env. Viral particles produced from CasES+4070A-NSCS contained both CasBrE and 4070A gp70 Env proteins. These particles displayed ecotropic receptor-mediated infection, but were still 100-fold less efficient than CasE+4070A-NSC virus. Infectious center analysis showed CasBrE SU ecotropic transduction efficiencies approaching those of NSCs expressing full length CasBrE Env (CasE; SU+TM). In addition, CasBrE SU-4070A Env interactions resulted in robust ecotropic superinfection interference indicating near native intracellular SU interaction with its receptor, mCAT-1.ConclusionsIn this report we provided evidence that 4070A Env and CasBrE SU physically interact within NSCs leading to CasBrE SU retention on the plasma membrane, incorporation into viral particles, restoration of mCAT-1 binding, and capacity for initiation of TM-mediated fusion events. Thus, heterotropic Env-SU interactions facilitates CasBrE SU folding events that restore Env activity. These findings are consistent with the idea that one protein conformation acts as a folding scaffold or nucleus for a second protein of similar primary structure, a process reminiscent of prion formation. The implication is that template-based protein folding may represent an inherent feature of neuropathogenic proteins that extends to retroviral Envs.

Mapping the folding pathway of the transmembrane protein DsbB by protein engineering

The four-helical transmembrane protein DsbB (disulfide bond reducing protein B) folds and unfolds reversibly in mixed anionic/non-ionic micelles, consisting of an unfolding intermediate I and a rate-limiting transition state (TS) between I and the denatured state D. Here, I describe the analysis of the folding behavior of 12 different alanine-scanning mutants of DsbB. For all mutants, TS is as compact as D and there is an accelerating increase in compaction as the protein proceeds to I and the native state. This unusual pattern of consolidation may reflect significant amounts of secondary structure in D, analogous to a classical folding intermediate. Unexpectedly, an increase in apolar surface area upon mutation is stabilizing whereas an increase in polar surface area is destabilizing. This effect is probably dominated by the effect of the mutations on the structure of the denatured state. I observe clear Hammond postulate behavior, in which a destabilization of I moves it closer to D. Φ-Value analysis indicates that in TS, a folding nucleus consisting of two to three residues with Φ-values of > 0.5 forms at one end of the transmembrane helices, which expands to include residues closer to the middle of the protein in I. Thus, folding proceeds from a highly polarized starting point.

Folding Catalysis by Transient Coordination of Zn2+ to the Cu Ligands of the ALS-Associated Enzyme Cu/Zn Superoxide Dismutase 1

How coordination of metal ions modulates protein structures is not only important for elucidating biological function but has also emerged as a key determinant in protein turnover and protein-misfolding diseases. In this study, we show that the coordination of Zn(2+) to the ALS-associated enzyme Cu/Zn superoxide dismutase (SOD1) is directly controlled by the protein's folding pathway. Zn(2+) first catalyzes the folding reaction by coordinating transiently to the Cu ligands of SOD1, which are all contained within the folding nucleus. Then, after the global folding transition has commenced, the Zn(2+) ion transfers to the higher affinity Zn site, which structures only very late in the folding process. Here it remains dynamically coordinated with an off rate of ∼10(-5) s(-1). This relatively rapid equilibration of metals in and out of the SOD1 structure provides a simple explanation for how the exceptionally long lifetime, >100 years, of holoSOD1 is still compatible with cellular turnover: if a dissociated Zn(2+) ion is prevented from rebinding to the SOD1 structure then the lifetime of the protein is reduced to a just a few hours.

Hydrophobic Core Formation and Dehydration in Protein Folding Studied by Generalized-Ensemble Simulations

Despite its small size, chicken villin headpiece subdomain HP36 folds into the native structure with a stable hydrophobic core within several microseconds. How such a small protein keeps up its conformational stability and fast folding in solution is an important issue for understanding molecular mechanisms of protein folding. In this study, we performed multicanonical replica-exchange simulations of HP36 in explicit water, starting from a fully extended conformation. We observed at least five events of HP36 folding into nativelike conformations. The smallest backbone root mean-square deviation from the crystal structure was 1.1 Å. In the nativelike conformations, the stably formed hydrophobic core was fully dehydrated. Statistical analyses of the simulation trajectories show the following sequential events in folding of HP36: 1), Helix 3 is formed at the earliest stage; 2), the backbone and the side chains near the loop between Helices 2 and 3 take nativelike conformations; and 3), the side-chain packing at the hydrophobic core and the dehydration of the core side chains take place simultaneously at the later stage of folding. This sequence suggests that the initial folding nucleus is not necessarily the same as the hydrophobic core, consistent with a recent experimental ϕ-value analysis.

SH3 Domain Unfolding Pathway Studied by Protein Contact Network Model

The approach of complex networks has provided one powerful tool to understand how proteins consistently fold into their native-state structures and the relevance of structure to their function. In this study, a coarse-grained complex network model called Protein Contact Network (PCN) is employed to study the unfolding process of SH3 domain, and the change of the network parameters associated with the conformational changes was analyzed on the SH3 unfolding pathway. The results show that the average clustering coefficient is less sensitive to the structural change, however the average shortest path lengths can examine the larger structural changes. Betweenness parameters, which characterize the geometrical properties of hydrophobic cores and folding nucleus residues, are shown out by the significant maximum values of native and transition state in the networks, respectively. It is easy to distinguish the folding nucleus from other residues by the betweenness of the transition state network, and it is easy to identify the hydrophobic core regions with the betweenness of the native state network. The results indicate that the unfolding of SH3 is mainly exhibited as the decrease of the long-rang links, and both the hydrophobic core and folding nucleus of the protein become derogated from its native state to the denature state. The hydrophobic collapse model and nucleation condensation model become reconciled in this work.

A COMPARATIVE ANALYSIS OF FOLDING PATHWAYS OF THERMOPHILIC AND MESOPHILIC PROTEINS BY MONTE CARLO SIMULATIONS

In this work we have studied the folding pathways for four pairs of homologous proteins from thermophilic and mesophilic organisms from two different structural classes (class a, all-alpha proteins and class d, alpha + beta proteins) using Monte Carlo simulations. We have obtained 50 trajectories for each protein and followed the free-energy profile and the order of folding of secondary structure elements between the last occurrence of the completely unfolded state and the first occurrence of the completely folded state. It turns out that the period of successful crossing of the free-energy barrier between unfolded and folded states for 40-45 trajectories (80-90%) makes 10% of the total folding time for four proteins (1tzvA, 1eyvA, 351c, and 1t4aA) and only 0.1% for two proteins (1dd3, 1ctf). This observation can be explained by a higher free-energy barrier for these proteins (1dd3, 1ctf) in comparison with other studied proteins. We have observed that folding pathways of thermophilic and mesophilic proteins may be the same, partly the same, and different. And similarity or difference between the folding pathways of thermophilic and mesophilic proteins does not depend on the structural class to which these proteins belong. Folding pathways for proteins from both classes correlate with the calculated folding nuclei for these proteins.

Protein Folding as Flow across a Network of Folding−Unfolding Pathways. 2. The “In-Water” Case

This paper, second in the series, extends our analysis of networks of protein folding-unfolding pathways in vitro from the equilibrium, analytically the simplest, to "in-water" conditions, i.e., to the case when the native state of a protein is, as a rule, much more stable than the unfolded one. Protein folding rates and folding nuclei determined for such "physiological" conditions are of special biological and medical interest because of their relevance to misfolding diseases and other folding-related disorders. Given the native protein structures and their experimental in-water stabilities, the previously developed general theory (see paper 1 of this series) is applied here to compute the in-water folding and unfolding rates and outline the folding nuclei. Agreement between these calculations and experiment appears to be reasonably good.

Protein Folding as Flow across a Network of Folding−Unfolding Pathways. 1. The Mid-Transition Case

Prediction of protein folding rates and folding nuclei is an important problem of protein science. Most of the previously proposed models for protein folding in vitro are based on the nucleation mechanism of this process. Our model considering protein folding as a flow arising in a network of folding-unfolding pathways at a coarse-grained free-energy landscape was described a few years ago, along with an algorithm for calculation of protein folding rates. Here we extend our approach and describe in detail a mathematically strict algorithm for calculating the "folding nuclei", arising as bottlenecks of the flow. Although the proposed physical theory uses no adjustable parameters, its results are in good agreement with experiment. This paper presents (i) the general theory and (ii) the results for the simplest case, i.e., folding/unfolding at the midpoint of thermodynamic equilibrium between the native and unfolded states of a protein; results for "in-water" conditions, i.e., for the case when no denaturant is added and the native state of a protein is much more stable than the unfolded one, will be described in the next paper of the series.

Kinetics and Reaction Coordinates of the Reassembly of Protein Fragments Via Forward Flux Sampling

We studied the mechanism of the reassembly and folding process of two fragments of a split lattice protein by using forward flux sampling (FFS). Our results confirmed previous thermodynamics and kinetics analyses that suggested that the disruption of the critical core (of an unsplit protein that folds by a nucleation mechanism) plays a key role in the reassembly mechanism of the split system. For several split systems derived from a parent 48-mer model, we estimated the reaction coordinates in terms of collective variables by using the FFS least-square estimation method and found that the reassembly transition is best described by a combination of the total number of native contacts, the number of interchain native contacts, and the total conformational energy of the split system. We also analyzed the transition path ensemble obtained from FFS simulations using the estimated reaction coordinates as order parameters to identify the microscopic features that differentiate the reassembly of the different split systems studied. We found that in the fastest folding split system, a balanced distribution of the original-core amino acids (of the unsplit system) between protein fragments propitiates interchain interactions at early stages of the folding process. Only this system exhibits a different reassembly mechanism from that of the unsplit protein, involving the formation of a different folding nucleus. In the slowest folding system, the concentration of the folding nucleus in one fragment causes its early prefolding, whereas the second fragment tends to remain as a detached random coil. We also show that the reassembly rate can be either increased or decreased by tuning interchain cooperativeness via the introduction of a single point mutation that either strengthens or weakens one of the native interchain contacts (prevalent in the transition state ensemble).

Direct Observation of Nucleation and Growth in Amyloid Self-Assembly

Access to native protein structure depends on precise polypeptide folding and assembly pathways. Identifying folding missteps that may lead to the nearly 40 protein misfolding diseases could feature prominently in the development of intervention strategies. Accordingly, we have investigated the earliest steps of assembly by the folding nucleus of the Alzheimer's disease Abeta peptide with real-time imaging and fluorescence correlation spectroscopy. These analyses reveal the immediate formation of large micrometer size clusters maintaining properties of intermolecular molten globules. These dynamic unstructured aggregates serve as the nucleating sites for amyloid growth and, as with native protein folding, appear important for backbone desolvation. The resulting amyloid nucleus however is able to template monomer addition from solution at rates from 2K peptides/s at millimolar peptide concentrations. This direct observation of amyloid assembly unifies several divergent models that currently exist for protein misfolding.

Folding Intermediate and Folding Nucleus for I→N and U→I→N Transitions in Apomyoglobin: Contributions by Conserved and Nonconserved Residues

Kinetic investigation on the wild-type apomyoglobin and its 12 mutants with substitutions of hydrophobic residues by Ala was performed using stopped-flow fluorescence. Characteristics of the kinetic intermediate I and the folding nucleus were derived solely from kinetic data, namely, the slow-phase folding rate constants and the burst-phase amplitudes of Trp fluorescence intensity. This allowed us to pioneer the ϕ-analysis for apomyoglobin. As shown, these mutations drastically destabilized the native state N and produced minor (for conserved residues of G, H helices) or even negligible (for nonconserved residues of B, C, D, E helices) destabilizing effect on the state I. On the other hand, conserved residues of A, G, H helices made a smaller contribution to stability of the folding nucleus at the rate-limiting I→N transition than nonconserved residues of B, D, E helices. Thus, conserved side chains of the A-, G-, H-residues become involved in the folding nucleus before crossing the main barrier, whereas nonconserved side chains of the B-, D-, E-residues join the nucleus in the course of the I→N transition.

Prediction of Stability upon Point Mutation in the Context of the Folding Nucleus

Proteins come in all shapes and sizes. Although it is possible to predict with reasonable success their structure from their sequence, the process of folding a chain of amino acids into its tertiary structure remains partially understood. This article addresses several characteristics pertaining to protein folding. The development of the Most Interacting Residues (MIR) algorithm, which dynamically simulates the early folding events, permits a reasonable ab initio prediction of the deeply buried critical residues involved in the formation of the protein core. The analysis of MIR positions with respect to protein 3D topology, in particular, to fragments called Tightened End Fragments (TEF) that might be good candidate for autonomous folding units, suggests that they are also essential for defining core stability. To validate this hypothesis, this study measures the sensitivity of MIR residues to point mutations. It is performed on a set of 385 proteins from a database that contains stability data calculated with five different algorithms. Tools have been developed to help the analysis and a consensus of the five methods is proposed. It results that positions predicted both as a MIR and a minimum of stability for the consensus are good candidates for the folding nucleus, and consequently their mutations may be hazardous.

Folding Study of Venus Reveals a Strong Ion Dependence of Its Yellow Fluorescence under Mildly Acidic Conditions

Venus is a yellow fluorescent protein that has been developed for its fast chromophore maturation rate and bright yellow fluorescence that is relatively insensitive to changes in pH and ion concentrations. Here, we present a detailed study of the stability and folding of Venus in the pH range from 6.0 to 8.0 using chemical denaturants and a variety of spectroscopic probes. By following hydrogen-deuterium exchange of (15)N-labeled Venus using NMR spectroscopy over 13 months, residue-specific free energies of unfolding of some highly protected amide groups have been determined. Exchange rates of less than one per year are observed for some amide groups. A super-stable core is identified for Venus and compared with that previously reported for green fluorescent protein. These results are discussed in terms of the stability and folding of fluorescent proteins. Under mildly acidic conditions, we show that Venus undergoes a drastic decrease in yellow fluorescence at relatively low concentrations of guanidinium chloride. A detailed study of this effect establishes that it is due to pH-dependent, nonspecific interactions of ions with the protein. In contrast to previous studies on enhanced green fluorescence protein variant S65T/T203Y, which showed a specific halide ion-binding site, NMR chemical shift mapping shows no evidence for specific ion binding. Instead, chemical shift perturbations are observed for many residues primarily located in both lids of the beta-barrel structure, which suggests that small scale structural rearrangements occur on increasing ionic strength under mildly acidic conditions and that these are propagated to the chromophore resulting in fluorescence quenching.

Two-state protein-like folding of a homopolymer chain

Many small proteins fold via a first-order “all-or-none” transition directly from an expanded coil to a compact native state. Here we study an analogous direct freezing transition from an expanded coil to a compact crystallite for a simple flexible homopolymer. Wang-Landau sampling is used to construct the 1D density of states for square-well chains of length 128. Analysis within both the micro-canonical and canonical ensembles shows that, for a chain with sufficiently short-range interactions, the usual polymer collapse transition is preempted by a direct freezing or “folding” transition. A 2D free-energy landscape, built via subsequent multi-canonical sampling, reveals a dominant folding pathway over a single free-energy barrier. This barrier separates a high entropy ensemble of unfolded states from a low entropy set of crystallite states and the transition proceeds via the formation of a transition-state folding nucleus. Despite the non-unique homopolymer ground state, the thermodynamics of this direct freezing transition are identical to the thermodynamics of two-state protein folding. The model chain satisfies the van’t Hoff calorimetric criterion for two-state folding and an Arrhenius analysis of the folding/unfolding free energy barrier yields a Chevron plot characteristic of small proteins.

Residue Specific Analysis of Frustration in Folding Landscape of Repeat Alpha/Beta Protein Apoflavodoxin

Topological frustrated proteins can give rise to complex folding pathways and transition state profiles. To better understand such systems, we combine experimental and computational methods to study Desulfovibrio desulfuricans apoflavodoxin by producing several point mutation variants. By equilibrium unfolding experiments, we first revealed how different secondary-structure elements contribute to overall protein resistance towards heat and urea. Next using stopped-flow mixing coupled to far-UV circular dichroism (CD), we probed how individual residues affect the amount of structure formed in the experimentally-detected burst-phase intermediate. Together with in silico folding route analysis of the same point-mutated variants and computation of the growth in nucleation size during early folding, computer simulations suggested the presence of two competing folding nuclei at opposite sides of the central β-strand 3 (i.e. at β-strands 1 and 4), which cause early topological frustration (i.e., misfolding) in the folding landscape. Particularly, the extent of heterogeneity in the folding nucleigrowth correlates with the in vitro burst phase CD amplitude. In addition, Φ-value analysis (in vitro and in silico) of the overall folding barrier to apoflavodoxin's native state revealed that native-like interactions in most of the β-strands must form in transition state. Our study reveals that an imbalanced competition between the two sides of apoflavodoxin's central β-sheet directs initial misfolding while proper alignment on both sides of β-strand 3 is necessary for productive folding.

Multiple Evolutionary Mechanisms Reduce Protein Aggregation

The folding of polypeptides into stable globular protein structures requires protein sequences with a relatively high hydrophobicity and secondary structure propensity. These biophysical properties, however, also favor protein aggregation via the formation of intermolecular beta-sheets and, as a result, globular structure and aggregation are inextricable properties of protein polypeptides. Aggregates that are enriched in beta-sheet structures have been found in diseased tissues in association with at least twenty different human disorders and the effect of aggregation on protein function include simple loss-of-function but also often a gain of toxicity. Given both the ubiquity and the potentially lethal consequences of protein aggregation, negative selective pressure strongly minimizes aggregation. Various evolutionary strategies keep aggregation in check, including (1) the optimisation of the thermodynamic stability of the protein, which precludes aggregation by burial of the aggregation prone regions in solvent inaccessible regions of the structure, (2) segregation between folding nuclei and aggregation nuclei within a protein sequence, (3) the placement of so-called gatekeeper residues at the flanks of aggregating segments, that reduce the aggregation rate of (partially) unfolded proteins, and (4) molecular chaperones that target aggregation nucleating sequences directly, thereby further suppressing aggregation in a cellular environment. In this review we describe the intrinsic features built into protein sequence and structure that protect against aggregation.

The HD-exchange motions of ribosomal protein S6 are insensitive to reversal of the protein-folding pathway

An increasing number of protein structures are found to encompass multiple folding nuclei, allowing their structures to be formed by several competing pathways. A typical example is the ribosomal protein S6, which comprises two folding nuclei (sigma1 and sigma2) defining two competing pathways in the folding energy landscape: sigma1 --> sigma2 and sigma2 --> sigma1. The balance between the two pathways, and thus the order of folding events, is easily controlled by circular permutation. In this study, we make use of this ability to manipulate the folding pathway to demonstrate that the dynamic motions of the S6 structure are independent of how the protein folds. The HD-exchange protection factors remain the same upon complete reversal of the folding order. The phenomenon arises because the HD-exchange motions and the high-energy excitations controlling the folding pathway occur at separated free-energy levels: the Boltzmann distribution of unproductive unfolding attempts samples all unfolding channels in parallel, even those that end up in excessively high barriers. Accordingly, the findings provide a simple rationale for how to interpret native-state dynamics without the need to invoke fluctuations off the normal unfolding reaction coordinate.